The physiological pathway which is involved in hemostasis and tissue repair is initiated by the release of thromboplastin from injured cells. Upon contact with factor VII in the surrounding plasma, factor X activator is formed. Together with factor V, along with associated phospholipids and calcium, prothrombin is converted into thrombin. The enzymatic activity of thrombin results in cleavage of fibrinogen to form fibrin monomers, which aggregate and are covalently crosslinked via the activity of factor XIIIa (which is formed via thrombin activation of factor XIII). See FIG. 1.
In order to promote rapid hemostasis and wound healing, many clinicians have focused on the development of hemostatic compositions which include various coagulation factors and function by exploiting the body's own hemostatic processes. For example, fibrin powder has been used as a hemostatic agent for many years. In the mid 1900s, surgeons began to use fibrin-containing preparations as in situ fibrin polymerizing agents. To initiate fibrin clot formation in such compositions, concentrated plasma and ground tissue (muscle or lung) were often used as the as the catalyst for peripheral nerve anastomosis (Young, et al., Lancet 2:126-8, 1940.) In 1944, Tidrick et al. utilized human plasma and bovine thrombin in solution as a two-part sealant to attach skin grafts (Tidrick, et al., Surgery 15:90-95 (1944)). The use of plasma and thrombin for these and other applications soon fell out of favor due to premature failure of the bond. This was attributed to low mechanical strength, which was in turn attributed to low fibrinogen concentration.
Fibrin sealants utilizing concentrated fibrinogen in the form of homologous cryoprecipitate were reported by Matras, et al. in Oral Maxillofac. Surg. 43:605-611 (1985). These types of fibrin sealants are commercially available under the name Tisseel.RTM. (Immuno AG, Vienna, Austria) and Beriplast P.RTM. (Centeon AG, Germany), and are reviewed in Sierra, J Biomater. Appl., 7:309-352 (1993). However, these types of fibrin sealants generally involve the administration of pooled blood products.
In an effort to avoid the problems associated with administration of pooled blood products, some investigators turned to the use of tissue sealant compositions utilizing patient autologous plasma as a source of fibrinogen, which was then applied in combination with bovine topical thrombin in calcium chloride solution (see, e.g., Siedentop, et al., Laryngoscope 95: 1074-1076 (1985)). However, performance of these materials was somewhat limited because of their inferiority in comparison to the ready-to-use homologous products. In addition, the use of these materials is limited due to performance and method of use problems associated with the variability of properties of the autologous plasma.
These problems have led to the development of hemostatic compositions which are based on the use of polymeric materials as adhesives. For example, synthetic polymerizable compositions such as cyanoacrylates have been used in adhesive compositions (Ellis, et al., J. Otolaryngol. 19:68-72 (1990)). However, the toxicity of many of these synthetic polymer compositions has limited their usefulness.
Natural substances can also exhibit bonding characteristics. In particular, collagen has been reported as being useful as a hemostatic agent (U.S. Pat. No. 4,215,203.) However, when compared with either collagen or synthetic polymers, adhesives which depend on fibrin exhibit enhanced hemostasis (Raccula, et al., Am. J Surg. 163 (2): 234-238 (1992)).
Thrombin-based formulations for use as hemostatics have also previously been described. See, for example, U.S. Pat. Nos. 2,433,299 and 4,363,319. However, the use of thrombin in hemostatic formulations is limited by its instability during storage. Accordingly, many investigators have turned to the use of thrombin in dry form, or its use in conjunction with a substrate such as a solid matrix. In particular, U.S. Pat. No. 4,515,637 describes the use of thrombin and collagen in the formation of sponges for use in treating wounds. Additionally, U.S. Pat. No. 5,464,471 describes the formation of dry thrombin formulations which are used in conjunction with fibrin monomers.
Recent developments have also led to the production of thrombin-fibrin compositions, which are formulated and used either as "dual component" compositions, or "single component" compositions. In either case, such compositions are sometimes referred to as "fibrin glue", with the thrombin functioning as the "catalyst" component of the glue, and the fibrin functioning as the "resin" component of the glue. Dual-component compositions generally involve the use of separate fibrinogen-containing and thrombin-containing components which are mixed together shortly before or simultaneously upon administration. See, for example, U.S. Pat. No. 5,290,552, which describes a dual-component composition that may optionally contain collagen in the fibrinogen-containing component. Single-component compositions generally include both a source of thrombin and a source of fibrin.
In either the single-or dual-component compositions, the fibrin is usually supplied in the form of fibrinogen, which is then converted to fibrin by thrombin. Although the single-component systems are more convenient to use, these compositions generally require the thrombin to be maintained in an inactive state prior to use to prevent premature coagulation. This problem has been addressed in the following two U.S. Patents: U.S. Pat. No. 5,318,524 describes the development of single-component hemostatic compositions containing thrombin which has either been inactivated or physically separated (using different phases) from fibrinogen prior to use; and U.S. Pat. No. 5,407,671 describes a single-component composition containing a thrombin inhibitor along with the thrombin.
The present invention relates to thrombin-based hemostatic compositions which can reproducibly and effectively be used as a single-component, or in conjunction with a source of fibrinogen, such as autologous plasma. By employing microfibrillar collagen as an additional component in the compositions, many of the aforementioned shortcomings of other hemostatic agents can be avoided.